Human cell populations typically have a finite lifetime, dividing a number of times before entering a nondividing phase called “replicative senescence”. Human chromosomes are capped with repeated sequences called “telomeres”. Human telomeres consist of up to about 2500 repeats of the sequence 5′-TTAGGG-3′ (SEQ ID NO:1). Telomeres in normal non-cancerous cells shorten each time that a cell divides. This has been viewed as a type of ‘clock’, helping to determine the lifespan of a cell population.
Several types of cells are “immortal”. These include germline cells, unicellular eukaryotes, and certain cancer cells. These types of cells are able to maintain the length of their telomeres by the use of an enzyme. Telomerase is an enzyme that catalyzes the addition of a specific repeating DNA sequence onto the 3′ end of DNA strands, thereby lengthening the telomere. The telomerase activity allows cells to compensate for telomere shortening that occurs during DNA replication. Telomerase activity is not detected in most normal somatic cells.
A connection between telomeres and aging was established by Bodnar et al. (Science, 279: 349-352, 1998). Transfection of a telomerase-encoding vector into telomerase-negative retinal pigment epithelial cells and foreskin fibroblasts showed dramatic effects. While non-transfected cells exhibited typical telomere shortening and senescence, the transfected cells showed lengthened telomeres and reduced levels of β-galactosidase (a marker for senescence). Furthermore, the transfected cells were reported to exceed their normal lifespan by at least 20 doublings.
Nakamura et al. (Science 277: 955-959, 1997) reported the isolation and sequencing of the human telomerase reverse transcriptase (hTERT) gene. Expression of hTERT mRNA corresponded with telomerase activity in cells. Meyerson et al. (Cell 90: 785-795, 1997) reported that hTERT (hEST2) is expressed at high levels in primary tumors, cancer cell lines, and telomerase-positive tissues but is undetectable in telomerase-negative cell lines and differentiated telomerase-negative tissues. Introduction of a nucleic acid sequence encoding the hTERT protein into mortal cells was shown to produce active telomerase (Weinrich, S. L., et al., Nat. Genet. 17(4): 498-502, 1997).
Interestingly, telomeres can be maintained in certain human cells that lack telomerase. An alternative pathway involving recombination has been found to be operative in several types of eukaryotes. In addition, a rolling circle mechanism has been suggested as providing a different alternative biological pathway for elongation of telomere repeat length (McEachern, M. J. et al., Annu. Rev. Genet. 34(1): 331-358, 2000). Extrachromosomal telomeric DNAs that may be present in circular form have been reported to be isolated from eukaryotic cells (Regev, A. et al., Oncogene 17: 3455-3461, 1998). However, this mechanism has not yet been proven, and it is not clear how such large double-stranded circles could initiate telomere extension, nor is it clear how such double stranded circles containing only telomere sequences could be synthesized.
The understanding of telomerase activity has led to two prospective applications: to increase the lifespan of non-immortal cells, and to kill cancerous immortal cells. Attempts to achieve both results have traditionally focused on upregulating or downregulating the telomerase enzyme itself.
Recently, attention has been turned towards potential chemical methods for achieving either goal (Cech, T. R., Angew. Chem. Int. Ed. Engl. 39(1): 34-43, 2000). Chemicals which regulate telomerase activity could be used as a potential cancer therapeutic or for increasing the replicative lifespan of a normal cell population. However, to date no such chemicals have been reported.
“Rolling circle” DNA replication is based on the observation that small circular DNA molecules act as efficient templates for DNA polymerases (Fire, A. and Xu, S. Q., Proc. Natl. Acad. Sci. USA. 92(10): 4641-4645, 1995; Liu, D. Y. et al., J. Am. Chem. Soc. 118(7): 1587-1594, 1996). Rolling circle replication had previously been reported in bacteriophage DNA amplification (Nakai, H., J. Biol. Chem. 268(32): 23997-4004, 1993).
Rolling circle transcription has further been used, with RNA polymerases, to produce catalytic RNAs and circular RNAs (Sarah L. Daubendiek, S. L. and Kool, E. T., Nature Biotech. 15: 273-277, 1997; Diegelman, A. M. and Kool, E. T., Nucleic Acids Res. 26: 3235-3241, 1998; Daubendiek, S. L. et al., J. Am. Chem. Soc. 117: 7818-7819, 1995).
Rolling circle amplification (RCA) has been used in vitro for the linear or geometric amplification of circular oligonucleotide probes (Andras, S. C. et al., Mol. Biotechnol. 19(1): 29-44, 2001). Recently, rolling circles have been reported as being useful for detecting gene copy number and point mutations in fixed cells (Christian, A. T. et al., Proc. Natl. Acad. Sci. U.S.A. 98(25): 14238-14243, 2001).
U.S. Pat. No. 6,096,880 (issued Aug. 1, 2000) and U.S. Pat. No. 5,714,320 (issued Feb. 3, 1998) describe the use of a single stranded circular oligonucleotide template and an RNA polymerase to produce an RNA multimer. The multimers are suggested as being useful for diagnostic and/or therapeutic applications. U.S. Pat. No. 6,077,668 (issued Jun. 20, 2000) describes the labeling of such multimers and their use in diagnostic applications. The above multimers do not use primers, and the circles do not contain repeating sequences. U.S. Pat. No. 5,872,105 (issued Feb. 16, 1999) describes single stranded DNA circles, and their potential uses. The circles bind both single- and double-stranded DNA molecules, and have potential use as a drug delivery device. Circular DNA molecules have further been shown to form triplex structures upon binding to a target sequence (U.S. Pat. No. 5,683,874, issued Nov. 4, 1997). However, no circles encoding telomeric DNA sequences were contemplated, and methods for making such repeating-sequence circles were not known. Standard methods for synthesis of DNA circles are not successful for circles containing repeating sequences.
The telomerase enzyme and telomere elongation has been proposed as a possible therapeutic method in: treating aging skin (Funk, W. D. et al., Exp. Cell Res. 258: 270-278, 2000), treating pancreatic cells (Funk, W. D. et al., J. Endocrinol. 166: 103-109, 2000), and in bone marrow transplantation (Lee, J. et al., Bone Marrow Transplant 24: 411-415, 1999; Engelhardt, M. et al., Blood 90: 182-193, 1997). However, permanent telomerase expression may lead to an elevated cancer risk (Hahn, W. C. et al., Nature 400: 464-468, 1999; Yaswen, P. et al., J. Mamm. Gland Biol. 6: 223-224, 2001).
Mutant telomeres have been discussed as a possible anticancer therapeutic (Kim, M. M. et al., Proc. Natl. Acad. Sci. U.S.A. 98: 7982-7987, 2001). In order to use a mutant telomerase in an anticancer role, the sequence encoding the wild type telomerase RNA would have to be interrupted (likely via a “knock-out”), and a sequence encoding a mutant telomerase RNA would have to be introduced into the cell. This is difficult, and likely impractical in a clinical setting.
Small nucleic acid circles containing telomere repeats from the organism Oxytricha have been reported for use in structural studies (Chen, J. et al., Chem. Commun. 22: 2686-2687, 2002; J. Am. Chem. Soc. 123: 12901, 2001). A “G-quadruplex” was used to effect ligation of linear molecules into circular DNA. The circles are too small to effect telomere extension, and do not contain human telomere sequences. No use with polymerases was contemplated, and the methods for making them would not succeed with larger circles containing human sequences.
Double-stranded plasmid DNAs containing telomeric sequence were studied recently with the organism Kluyveromyces lactis (Natarajan, S. and McEachern, M. J. Mol. Cell. Biol. 22: 4512-4521, 2002). Those circular plasmids were shown to cause the telomeres to become lengthened by combined mechanisms involving integration, recombination, and rolling circle replication. The telomeric sequence was only a fraction of the total sequence of the plasmids. No plasmids of purely telomeric sequence were contemplated, and no single stranded DNAs were contemplated. The methods provided would not allow for either of these to be constructed. DNA circles that contain non-telomeric sequence would in many cases be undesirable because they cause unnatural non-telomeric sequences to be included in the extended telomere. Double-stranded circles would in many cases be undesirable because they could not hybridize to a telomeric end without being unwound.
Despite the considerable research reported to date, there still exists a need for effective therapeutic compositions and methods. The use of telomerase to extend telomeres is expensive due to the costs involved in obtaining the enzyme. Therapy with telomerase is also a poor method for clinical uses, as overexpression and permanent expression of the enzyme has been associated with an elevated cancer risk, and gene therapy is still highly risky and largely unproven in humans. The engineering of a mutant telomerase to eliminate cancer is technically challenging and likely impractical. Compositions and methods are needed that are both cost- and clinically-effective.